Identifying Epigenetic, Chromatin, and Transcriptomic landscapes to Improve SCNT Development in an Animal Model

项目摘要 体细胞核移植（SCNT）允许创造基因相同的动物， 农业目的，是生产生物医学用基因工程动物的有力工具 用于治疗的患者特异性胚胎干细胞（SCNT-ESC）的应用和产生。但是，在这方面， 长期开发的低效率限制了其广泛使用和实际效用。根本 问题是卵子如何不完全地重新编程体细胞供体细胞核。的总体目标 目前的建议概述在两个具体目标：1）确定表观遗传，染色质，和转录组 景观在体外受精（IVF）胚胎，然后阐明如何SCNT胚胎偏离这个 开发范式; 2）建立和测试新的方法来纠正重编程缺陷， 促进植入前和植入后胚胎中SCNT发育。我们创造了伟大的， 为精心分期的IVF和SCNT牛胚胎提供高质量的开放染色质图谱， 单细胞开放染色质谱分析以阐明染色质中胚胎内和胚胎间异质性 重新编程这些地图使我们能够第一次询问， 在正常的植入前发育过程中，基因组开放和关闭，以及SCNT胚胎如何偏离 从这个范式。我们的初步数据清楚地表明，早期卵裂阶段的SCNT胚胎 正确建立开放染色质，并暗示EGA的重要贡献者，转录因子（TF） DUX是SCNT胚胎的主要缺陷。我们假设染色质重编程的失败 在植入前发育过程中，SCNT在植入后发育中表现出来， 使用TF表达或植入前筛查的干预将改善SCNT发育成功。目的 1包括三个分目标：确定偏离IVF的性别特异性SCNT开放染色质区域 胚胎，或表现出性别特异性; 1.2.鉴定SCNT胚胎中的体细胞记忆， 使用DNA甲基化分析进行重编程;以及1.3.确定SCNT之间的基因表达差异 和IVF囊胚，并评估它们是否偏向ICM或TE谱系。根据这些数据，我们将 使用生物信息学分析来鉴定另外的TF/调节子以用于进一步的机制解剖。在目标2中， 将测试三个次级目标：2.1.如果EGA的异位执行（使用DUX和其他TF）可以重新激活 培养的成纤维细胞中的EGA程序; 2.2.如果使用TF靶向激活SCNT胚胎中的EGA网络， 或TF组合（来自2.1）将改善重编程并促进其发展，以及2.3。如果单身- 早期的细胞活检和开放染色质分析可以预测IVF和IVF的发育结果， SCNT胚胎。这些目标将提高农业和生物医学动物的生产效率 模型和建立人类临床应用的策略和见解。

PROJECT SUMMARY Somatic cell nuclear transfer (SCNT) allows for the creation of genetically identical animals for agricultural purposes, and is a powerful tool for production of genetically engineered animals for biomedical applications and generation of patient-specific embryonic stem cells (SCNT-ESCs) for therapeutics. However, the low efficiency of full term development limits its wide-spread use and practical utility. The fundamental problem is how the egg incompletely reprograms a somatic donor nucleus. The overall objectives of the current proposal are outlined in the two Specific Aims : 1) Identify epigenetic, chromatin, and transcriptomic landscapes in in vitro fertilized (IVF) embryos and then elucidate how SCNT embryos deviate from this developmental paradigm; and 2) Establish and test new approaches to correct reprogramming defects and improve SCNT development both in preimplantation and postimplantation embryos. We have created high- quality open chromatin maps for carefully-staged IVF and SCNT bovine embryos and leveraged cutting-edge single-cell open chromatin profiling to clarify intra- and inter-embryo heterogeneity in chromatin reprogramming. These maps allow us to interrogate, for the first time, how specific regions of the genome open and close during normal preimplantation development, and how SCNT embryos deviate from this paradigm. Our preliminary data clearly indicate early cleavage staged SCNT embryos do not establish open chromatin properly, and implicate an important contributor to EGA, a transcription factor (TF) DUX, as a major deficiency in SCNT embryos. We hypothesize that failures in chromatin reprogramming during SCNT preimplantation development manifest later in postimplantation development, and that targeted intervention using TF expression or preimplantation screening will improve SCNT developmental success. Aim 1 consists of three subaims: 1.1. Determine sex-specific SCNT open chromatin regions that deviate from IVF embryos, or exhibit sex-specificity; 1.2. Identify somatic memory in SCNT embryos that is resistant to reprogramming using DNA methylation profiling; and 1.3. Identify gene expression differences between SCNT and IVF blastocysts and evaluate if they are biased towards ICM or TE lineages. Based on these data, we will use bioinformatic analyses to identify additional TFs/regulators for further mechanistic dissection. In Aim 2, three subaims will be tested: 2.1. if ectopic enforcement of EGA (using DUX and other TFs) can reactivate the EGA program in cultured fibroblasts; 2.2. If targeted activation of the EGA network in SCNT embryos using TF or TF combinations (from 2.1) would improve reprogramming and facilitate their development and 2.3. If single- cell biopsy and open chromatin profiling at early stages can predict developmental outcomes of both IVF and SCNT embryos. These goals will improve the efficiency of generation of agricultural and biomedical animal models and establish strategies and insights for human clinical applications.